EP1068294B1 - Mikrobielle stämme und verfahren für die herstellung von biomaterialien - Google Patents

Mikrobielle stämme und verfahren für die herstellung von biomaterialien Download PDF

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EP1068294B1
EP1068294B1 EP99914271A EP99914271A EP1068294B1 EP 1068294 B1 EP1068294 B1 EP 1068294B1 EP 99914271 A EP99914271 A EP 99914271A EP 99914271 A EP99914271 A EP 99914271A EP 1068294 B1 EP1068294 B1 EP 1068294B1
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bacterial strain
nuclease
gene
strain
strains
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EP1068294A1 (de
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Gjalt W. Huisman
Laura Luo
Daniel M. Horowitz
Tillman U. Gerngross
Oliver P. Peoples
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Yield10 Bioscience Inc
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Metabolix Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/08—Reducing the nucleic acid content
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • the present invention is generally in the field of genetically engineered bacterial systems for the enhanced production and recovery of polymers such as intracellular proteins, polyhydroxyalkanoate materials, and extracellular polysaccharides.
  • Microbial fermentations are used for the manufacture of a large number of pharmaceutical and industrial products, including antibiotics, organic acids, amino acids, proteins, vitamins, polymers such as polyhydroxyalkanoates, and polysaccharides ( Atkinson & Mavituna, "Biochemical Engineering and Biotechnology Handbook," 2nd edition, (Stockton Press, USA 1991 )).
  • Increased productivity and recovery of more highly purified product are major areas of development to increase profitability.
  • the industry trend generally is towards higher cell density fermentations to increase productivity. Cell densities in excess of 100 g/L are routinely achieved. Decreasing overall fermentation process costs by increasing the recovery of the product from the cell biomass or, in some cases, from the medium, is another means of increasing productivity.
  • PHAs Polyhydroxyalkanoates
  • a number of bacterial strains and fermentation processes have been described for the production of PHAs, such as described, for example, in Lee & Chang, Advances in Biochemical Engineering Biotechnology, 52:27-58 (1995 ); Poirier et al., Bio/Technology 13:142-50 (1995) ; Doi, Macromol. Symp. 98:585-99 (1995 ).
  • PHA production methods using specific bacterial strains are described in U.S. Patents No.
  • PHAs accumulate inside the microbial cells as discrete granular inclusions which although amorphous in nature are water insoluble.
  • Recovery of PHAs from the cells following fermentation can be accomplished by any of several methods, including (1) solvent extraction; (2) chemical destruction of all non-PHA biomass using hypochlorite, hydrogen peroxide or ozone treatment; and (3) milder processing involving cell disruption, enzyme treatment, and washing. In the latter process, it is possible to retain the PHA granules in the amorphous state and use the washed granule suspension as a latex. It is necessary to lyse PHA-containing cells in order to obtain a PHA latex from the cells using an aqueous process.
  • Fermentation processes are widely used for the manufacture of enzymes and other bioactive proteins.
  • recovery can be improved by the addition of nuclease to the crude cell lysates to degrade nucleic acid.
  • this approach also completely degrades DNA, which effectively eliminates the possible spread of antibiotic resistance markers or other genetic elements.
  • the process of adding exogenous nucleases is expensive.
  • Microbial polysaccharides are produced by fermentation of a number of different microorganisms. (Delest, P. 1989, pp. 301-313. Fermentation technology of microbial polysaccharides in Gums and Stabilisers for the Food Industry 5, Phillips, G.O., Wedlock, D.J. and Williams, P.A. eds. IRL Press at Oxford University Press, New York ).
  • Xanthomonas strains are used commercially for the production of xanthan gum ( Kennedy & Bradshaw, Prog. Ind. Microbiol. 19:319-71 (1984 )), and have been subjected to genetic engineering techniques ( Hassler & Doherty, Biotechnol. Prog.
  • a first aspect of the invention provides a bacterial strain producing a fermentation product selected from the group consisting of antibiotics, organic acids, amino acids, proteins, vitamins, polyhydroxyalkanoates, and polysaccharides, wherein the bacterial strain is genetically engineered to express a heterologous nuclease gene or a modified homologous nuclease gene or is mutated to cause enhanced heterologous or homologous nuclease gene product activity; and wherein the nuclease is secreted into the periplasm or a growth medium in which the bacterial strain is fermented in an amount effective to degrade at least 95% of all the nucleic acid released following lysis of the bacterial strain cells in less than 24 hours so that recovery of the product is enhanced.
  • a second aspect of the invention provides a fermentation process comprising adding to a growth medium the bacterial strain of the first aspect of the invention.
  • DNA constructs and genetically engineered microbial strains constructed using these DNA constructs which produce a heterologous or modified homologous nuclease enzyme with specificity for DNA and/or RNA, have been developed.
  • the heterologous nuclease can be obtained from another organism, engineered to increase enzyme activity, or homologous nucleases in the same strains mutated and then selected using assays for nuclease activity to select those bacteria expressing nucleases with enhanced activity.
  • These strains secrete nuclease into the periplasm or growth medium in an amount effective to enhance recovery of the polymers and are particularly suited for use in high cell density fermentation processes.
  • Exemplary polymers include intracellular proteins, such as enzymes, growth factors, and cytokines; polyhydroxyalkanoates; and polysaccharides, such as xanthan gum, alginates, gellan gum, zooglan, hyaluronic acid and microbial cellulose.
  • Methods have been demonstrated for improving the fermentation and recovery process for microbial fermentation products by using strains which secrete a nuclease during the fermentation process wherein the nuclease itself is not the desired fermentatation product.
  • Methods for genetically engineering strains to secrete nucleases useful in this method are disclosed.
  • a strain which does not express a suitable nuclease is genetically engineered to express an heterologous nuclease with sufficient activity to obtain the desired process improvement as defined in claim 1.
  • a strain which expresses a nuclease but at insuficient activity to obtain the desired process improvement is genetically modified to express sufficient nuclease using the isolated nuclease gene from that strain and the methods described herein.
  • a strain which expresses a suitable nuclease from a heterologous or homologous nuclease gene is mutated and mutants expressing sufficient levels as defined in claim 1 of nuclease selected to obtain the desired process improvement
  • enhance refers to an improved fermentation process wherein nucleic acid released by cell lysis during the fermentation is degraded by the nuclease, in an amount preventing viscosity increases, which can result in foaming, poor mixing and greater energy required for mixing.
  • enhanced also refers to an improved recovery process wherein, in a process in which the desired product is produced intracellularly and requires cell lysis for recovery, nucleic acid released by cell lysis during the fermentation is degraded following the cell lysis step, in an amount preventing viscosity increases and facilitating the recovery steps which may include centrifugation filtration, precipitation etc.
  • heterologous nuclease gene refers to a nuclease gene isolated from a host other than the strain to be improved.
  • homologous nuclease gene refers to a nuclease gene isolated from the host to be improved. This gene may be further modified to improve the activity of the nuclease.
  • nucleases and genes encoding nucleases which are suitable for use in the methods described herein can be obtained from a number of sources.
  • heterologous nucleases are expressed from nucleic acid encoding the nucleases, wherein the nuclease has been obtained from an organism other than the one in which it is inserted to enhance polymer production and/or recovery; or where the organism is mutated and screened for enhanced nuclease activity.
  • Enhanced nuclease activity can be obtained where the amount of nuclease, or the specific activity or substrate specificity of the nuclease, are increased.
  • Nucleases are secreted by a broad range of microorganisms and some of the corresponding genes have been cloned and characterized.
  • Sources of nuclease genes include S . marcescens (GenBank Acc. No. M19495), Anabaena PCC7120 (GenBank Acc. No. X64706), Bacillus subtilis (GenBank Acc. No. U66480), Staphylococcus hyicus (GenBank Acc. No. L23973), Staphylococcus intermedius (GenBank Acc. No. X67678), Escherichia coli (GenBank Acc. No. X55818), Shigella flexneri (GenBank Acc. No. U30471), Methanobacterium thermoautotrophicum (GenBank Acc. No. AE000833), and Methanococcus jannaschii (GenBank Acc. No. U67584).
  • the nuclease is cleaves both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the nuclease preferably is active over a wide temperature range and pH range.
  • the nuclease also preferably is tolerant to the presence of processing additives, such as salts, surfactants, and stabilizers. Following cell lysis and an incubation period to allow the nuclease to degrade the nucleic acid, it is preferable that the nuclease be readily removable, for example, by protease digestion, chromatography, filtration or centrifugation.
  • Micrococcal nuclease [(deoxy)ribonucleate-3'-nucleotidohydrolase, EC 3.1.4.7] can hydrolyze either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) to produce 3'-phosphomonunucleotides and dinucleotides.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the enzyme is stable at pH between about 0.1 and 10 with maximum activity around pH 9 to 10 in the presence of 0.1-100 mM Ca 2+ ions.
  • the gene encoding this enzyme has been isolated and characterized from Staphylococcus aureus ( Shortle, Gene 22:181-89 (1983 )) and is functionally expressed in a number of heterologous bacteria ( Shortle, Gene 22:181-89 (1983 ); Miller, et al., J. Bacteriol. 169:3508-14 (1987 ); Liebl, et al., J. Bacteriol. 174:1854-61 (1992 ); LeLoir, et al., J. Bacteriol. 176:5135-39 (1996 )).
  • nuclease gene encoding the selected nuclease enzyme is integrated into the microbial strain so that the organism will secrete nuclease into the periplasm or growth medium.
  • microbial strains for the production of short chain-length PHAs R eutropha, E. coil ) or medium chain-length PHAs ( Pseudomonas sp. MBX978 and P. putida ) are generated by integrating a nuclease gene, such as the S . aureus nuclease gene, into the chromosome of PHA producing strains.
  • a nuclease encoding gene can be isolated from S . aureus by amplification using the polymerase chain reaction with oligonucleotides that recognize the nuclease encoding nuc gene.
  • the amplified DNA can be cloned into the PCR cloning vector pCR2.1 (Invitrogen Corp., San Diego, CA). Plasmids containing the nuclease gene insert can be identified and analyzed for expression of nuclease using DNAse agar plates (Difco Laboratories, Detroit MI).
  • nuclease gene into the PHA production strain of choice for expression of the nuclease gene.
  • the gene can be put under the control of an Ralstonia promoter and inserted into a broad host range cloning vector, such as pLAFR3.
  • pLAFR3 broad host range cloning vector
  • bacterium from genera such as Aeromonas, Azotobacter, Burkholderia, Comamonas, Methylobacterium, Paracoccus, Pseudomonas, Rhizobium, and Zoogloea.
  • an expression cassette can be engineered for the nuclease such that it can be integrated into the chromosome of the appropriate host and expressed.
  • Suitable expression cassettes are described, for example, in Herrero et al., J. Bacteriol. 172:6557-67 (1990 ).
  • the nuclease gene and a marker gene, such as an antibiotic resistance-conferring gene, are cloned between the recognition sequences for a transposase in vectors such as the pUT and pLOF series (Herrero et al., J. Bacteriol. 172 :6557-67 (1990)).
  • pUT and pLOF plasmids can only be maintained in E. coli strains that permit replication of this plasmid by virtue of the presence of a ⁇ pir lysogen.
  • the nuclease-marker construct is introduced into bacterial cells by conjugation from E. coli ⁇ pir strains or by transformation or electroporation with plasmid DNA. In the absence of ⁇ pir, the plasmid is lost from the cell.
  • the DNA fragment enclosed by, and including, the recognition sequences for the transposase will be transferred to the chromosome.
  • Cells in which this event has occurred can be isolated by selection by plating the conjugation, transformation or electroporation mixture on solid growth media containing the antibiotic for which resistance is conferred by the construct. Colonies of cells growing on this medium are then screened for nuclease activity in vivo or in vitro. In vivo activity can be examined by plating the putative nuclease producers on DNase test agar plates using visualization with 1 N HCl or methyl green (Difco Laboratories, Detroit MI).
  • In vitro activity can be determined by incubating culture supernatant or the aqueous supernatant of chloroform treated cells with high molecular weight DNA such as chromosomal DNA, followed by resolution of the DNA on pH buffered-agarose gels.
  • the engineered strains can be used in a variety of microbial fermentations utilized in the manufacture of pharmaceutical and industrial products, including antibiotics, organic acids, amino acids, proteins, vitamins, polyhydroxyalkanoates, and polysaccharides ( Atkinson & Mavituna, "Biochemical Engineering and Biotechnology Handbook," 2nd edition, (Stockton Press, USA 1991 )).
  • the fermentation process includes cell densities in excess of 100 g/L.
  • the strains are particularly useful in processes in which the product must be recovered by cell lysis, and more particularly in processes that requires chromatography, crystallization, precipitation, centrifugation, and/or filtration separation processes.
  • the methods and strains described herein are used in fermentation processes for the production of PHAs, such as described, for example, in Lee & Chang, Advances in Biochemical Engineering Biotechnology, 52:27-58 (1995 ); Poirier et al., Bio/Technology 13:142-50 (1995 ); Doi, Macromol. Symp. 98:585-99 (1995 ); U.S. Patents No. 4,477,654 to Holmes et al ., No. 5,364,778 to Byrom , No. 5,266,470 to Senior et al ., No. 5,346,817 to Akiyama et al ., No. 4,336,334 to Powell et al ., No.
  • the methods and strains described herein are used in the production of a highly pure PHA latex, which can have many applications. Examples of these applications are described in PCT WO 91/13207 (PHA latex compositions for coating paper); GB 2 292 648 A (PHA latex in architectural coating formulations); PCT WO 96/00263 (PHA latex as food, especially cheese, coatings); PCT WO 92/09211 and U.S. Patent No. 5,229,158 to Yalpani (PHA granule compositions for use as dairy cream substitutes); PCT WO 92/09210 and U.S. Patent No.
  • PHAs 5,225,227 to Yalpani
  • PCT WO 96/17369 PHAs used in the production of cathode ray tube components. It generally is necessary to lyse PHA-containing cells in order to obtain a PHA latex from the cells using an aqueous process. Methods for lysing microbial cells are well known and include physical homogenization, ultrasound waves, enzyme and detergent treatment, and freeze/thaw cycling.
  • Microbial polysaccharides can be produced by fermentation of a number of different microorganisms.
  • Xanthomonas strains are used commercially for the production of xanthan gum ( Kennedy & Bradshaw, Prog. Ind. Microbiol. 19:319-71 (1984 )), and Pseudomonas elodea strains are used to produce gellan gum ( Kang, et al., Appl. Environ Micro. 43:1086-91 (1982 )).
  • alginates primarily from brown algae, which are used extensively in the food industry ( Wells in Extracellular Microbial Polysaccharides (Sanford &Laskin, eds.) pp. 299 (ACS, Washington, DC 1977 )). Alginates can also be produced by fermentation of Azotobacter strains ( Chen et al.,Appl. Environ. Micro. 49:543-46 (1985 )). Hyaluronic acid is another example of a polymer which is produced for biomedical applications using Streptococci ( Dougherty & van de Rijn, J. Biol. Chem. 269:169-75 (1994 )).
  • the methods and systems described herein may be particularly advantageous when used in conjunction with the cell lysis approach to xanthan gum recovery described by Pollock in US 5,354,671 and by Murofushi et al., in US 5,705,368 , and by Homma et al., in US 5,679,556 .
  • Suitable Xanthomonas strains for practising the disclosed method are described by Bauer et al., in US 4,400,467 and by Pollock et al., in US 5,472,870 .
  • gellan gum is one member of a family of structurally related exopolysaccharidfes known as "sphingans' ( Pollock, T.J. 1993, J. Gen. Microbiol. 139: 1939-1945 , Pollock et. al., in US 5,854,034 and references therein).
  • Gellan gum is produced by Pseudomonas elodea (American Type Culture Collection strain Number ATCC 31461) by processes described in U.S. Pat. Nos. 4,326,052 ; 4,377,636 ; 4,385,126 and 4,503,084 .
  • This strain and mutant derivatives with enhance gellan production characteristics can be genetically manipulated as described by Fialho et. al., 1991 Letters in Applied Microbiology 12: 85-87 and by Pollock et. al., in US 5,854,034 ).
  • mutants of this strain defficient in the accumulation ofPHB can also be used to practice the disclosed method and are available from The American Type Culture Collection as Strain Number ATCC 53967.
  • Hyaluronic acid is produced by Streptococcus bacteria, as described, for example, by Ellwood et. al., US 5,563,051 .
  • compositions and methods of preparation and use thereof described herein are further described by the following non-limiting examples.
  • Plasmid pNuc1 has been described in Liebl, et al., J. Bacteriol., 174:1854-61 (1992 )). The sequence of the nuc gene encoding micrococcal nuclease has been determined ( Shortle, Gene 22:181-89 (1983 )) and is accessible under GenBank Acc. No. J01785. The nuc gene was amplified using the purified plasmid pNuc1 and the polymerase chain reaction (PCR) with primers
  • the nuc gene was excised from plasmid pCR2.1-nuc using restriction enzymes EcoRl and Acc65I and inserted in the corresponding sites of pUC18NotI to obtain pUC18-nucI.
  • a promoterless kanamycin gene from plasmid pBGS18 Spratt et. al., 1986, Gene 41: 337-342 ; ATCC Accession Number 37437) was obtained by PCR amplification using the following primers:
  • pMNX-nuc The resulting plasmid, pMNX-nuc, was linearized with SmaI and ligated with a blunt ended EcoRI / Acc65I fragment containing a promoterless kanamycin gene from pMNX-kan.
  • Recombinant pMNX-nuc-kan plasmids were isolated based on their kanamycin resistance conferred by expression of the nuc-kan operon from the lac promoter.
  • the nuc-kan operon subsequently was excised as a NotI fragment and inserted in the corresponding site of pUTkan.
  • Plasmid pMUX-nuc-kan was subsequently used to integrate the promoterless nuc-kan operon randomly in the chromosomes of P. putida KT2442. Conjugation of the plasmid was achieved using E. coli S17-1 [pMUXnuc-kan].
  • Transgenic P . putida strains were selected on E2/10 mM octanoate/kanamycin/chloramphenicol plates. Out of 12,000 random integrants, 1500 colonies were replica plated to DNAse agar plates to select nuclease expressing clones. Thirty-five nuclease expressing clones were selected for in vitro analysis of nuclease levels in the culture supernatant and in the periplasm of the strain. For this purpose, cells were spun down and the supernatant containing the secreted nuclease was collected.
  • the cell pellet subsequently was resuspended in fresh medium, and 100 ⁇ l of chloroform was added to 500 ⁇ l suspension to release periplasmic nuclease.
  • Cell debris subsequently was spun down.
  • DNA gel electrophoresis was conducted with samples treated with benzonase (0.02 U), with culture supernatant of nuclease integrated statins, and with supernatant of P. putida KT2442. Nuclease activity in the secreted and periplasmic fractions of nine different P. putida nuc integrants on chromosomal DNA from P. putida KT2442 was present. Cell debris was subsequently removed by centrifugation.
  • nuclease The presence of nuclease was determined by incubating aliquots of the clarified supernatant with 4 ⁇ g of P. putida chromosomal DNA for one hour at 37°C. As controls, chromosomal DNA was incubated with commercially available BENZONASE TM , 0.02 units as a positive control or without nuclease as a negative control. Following digestion, the samples were analyzed by agarose gel electrophoresis (Sambrook et. al., 1989, Molecular Cloning, a Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Using this test, strains which express nuclease acivity at different levels and extracellular or periplasmic space or both were readily identified.
  • P. putida MBX918 These strains are called p . putida MBX918 through 926.
  • P. putida MBX924 secretes nuclease to both periplasm and the extracellular medium and has the nuc-kan operon integrated in the 23S rRNA encoding gene.
  • Transgenic R. eutropha 40124 strains were selected after conjugation of the host strain with E . coli S 17-1 [pMUX-nuc-kan] on PCT/1% glucose/naladixic acid/kanamycin plates. Ten random colonies were inoculated in PCT/1% glucose/naladixic acid/kanamycin liquid medium and grown for 20 hr. at 30 °C. Only the R. eutropha MBX917 culture produced an active nuclease that was localized in the periplasm of the transgenic strain.
  • Transgenic P . putida MBX978 strains were selected after conjugation of the host strain with E . coli S17-1 [pMUX-nuc-kan] on E2/10 mM octanoate/ kanamycin/naladixic acid plates and subsequently replica plated on DNAse agar plates with the appropriate antibiotics. A total of 50 nuclease-positive colonies were replica plated to E2/10 mM octanoate/kanamycin/ naladixic acid plates.
  • Nine isolates ( P. putida MBX979-987) subsequently were grown in liquid E2/10 mM octanoate/kanamycin/naladixic acid medium.
  • Table 1 PHA Production by MBX978 Derivatives with an Integrated Nuclease Gene Strain nuclease a OD 600 b td (hr) c PHA d MBX978 - 3.7 1.5 34.3 MBX979 +++++ 3.6 1.6 30.7 MBX981 + 3.4 1.4 32.9 MBX982 ++ 3.5 1.6 32.0 MBX984 ++++ 2.8 1.6 14.9 MBX985 +++ 3.8 1.4 33.2 a relative nuclease activity; b optical density of the culture at 600 nm at the end of the experiment; c doubling time of the strain; d percentage PHA of the bacterial cell dry weight
  • E . coli MBX247 strains were selected after conjugation of the host strain with E . coli S17-1 [pMUX-nuc-kan] on E2/10 mM octanoate/kanamycin/naladixic acid plates and subsequently replica plated on DNAse agar plates with the appropriate antibiotics. A total of 75 nuclease-positive colonies were replica plated to E2/10 mM octanoate/0.5% corn steep liquor/kanamycin/ naladixic acid plates. Nine isolates were subsequently grown in liquid R10/2% glucose/kanamycin/naladixic acid medium.
  • the nuclease gene was successfully integrated in the chromosome (MBX988-991).
  • the nuclease activities in the different transgenic strains were analyzed.
  • DNA gel electrophoresis was conducted with samples treated with periplasmic fractions of E. coli MBX247, E . coli MBX988 grown on R10 medium, E . coli MBX988 grown on LB medium, R. eutropha MBX917, R. eutropha 40124, Pseudomonas MBX985, Pseudomonas MBX978, P. putida MBX924, P. putida KT2442, and with molecular weight markers.
  • the nuclease activities were analyzed in the different transgenic strains using the chromosomal DNA digestion and gele electrophoresis assay as described in Example 2.
  • Example 6 Isolation of PHA Granule Suspensions from P . putida MBX978 and a Nuclease Expressing Derivative
  • P. putida MBX978 and P. putida MBX985 were grown to a cell density of 200 g/L in 20 L fed-batch cultures with octanoate as a carbon source. Following fermentation, each culture was supplemented with 1 mM CaCl 2 and adjusted to pH 8.5 with ammonium hydroxide. The cultures then were lysed using a high-pressure homogenizer operating at pressures varying from 8,000 to 20,000 psi. Each sample of lysate was incubated for one hour at room temperature, and then the viscosity was determined at 20 °C using a Brookfield LVF Viscometer (#1 spindle, 60 rpm).
  • lysates were prepared from wild-type culture supplemented with commercial nuclease (BENZONASE TM , 10 ⁇ L/L culture). Results for the viscosity determinations are given in Table 2 and show that integration of the nuclease gene as in MBX985 resulted in reduced viscosity of the lysate to levels similar to that obtained for the wild-type strain with added benzonase.

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Claims (25)

  1. Bakterienstamm, der ein Fermentationsprodukt erzeugt, das aus der Gruppe ausgewählt ist, die aus Antibiotika, organischen Säuren, Aminosäuren, Proteinen, Vitaminen, Polyhydroxyalkanoaten und Polysacchariden besteht,
    wobei der Bakterienstamm gentechnisch so manipuliert ist, dass er ein Produkt eines heterologen Nuclease-Gens oder eines modifizierten homologen Nuclease-Gens exprimiert, oder so mutiert ist, dass eine erhöhte Aktivität eines Produkts eines heterologen oder homologen Nuclease-Gens bewirkt wird, und wobei die Nuclease in das Periplasma oder ein Wachstumsmedium, in dem der Bakterienstamm fermentiert wird, in einer Menge sezerniert wird, die bewirkt, dass wenigstens 95 % aller Nucleinsäuren, die nach der Lyse der Zellen des Bakterienstamms freigesetzt werden, in weniger als 24 Stunden abgebaut werden, so dass die Ausbeute des Produkts verbessert wird.
  2. Bakterium nach Anspruch 1, das in der Lage ist, bis zu Zelldichten von wenigstens 50 g/I zu wachsen.
  3. Bakterienstamm nach Anspruch 1 oder 2, der ein Polyhydroxyalkanoat erzeugt, vorzugsweise in Mengen von wenigstens 40 % seines Trockenzellgewichts.
  4. Bakterienstamm nach Anspruch 2 für den Einsatz in einem wässrigen Prozess zur Herstellung einer Suspension von Poly(3-hydroxyalkanoat)-Körnchen, die im Wesentlichen frei von Nucleinsäuren ist.
  5. Bakterienstamm nach Anspruch 1 für den Einsatz in einem Prozess zur Herstellung von Polysacchariden, die aus der Gruppe ausgewählt sind, die aus Xanthangummi, Alginaten, Gellangummi, Zooglan, Hyaluronsäure und mikrobieller Cellulose besteht.
  6. Bakterienstamm nach Anspruch 1, wobei das Nuclease-Gen ein heterologes Gen ist, das aus einem anderen Organismus als dem Bakterienstamm erhalten wurde.
  7. Bakterienstamm nach Anspruch 1, wobei das Nuclease-Gen in einen Wirtsstamm integriert ist, der aus der Gruppe ausgewählt ist, die aus Ralstonia eutropha, Methylobacterium organophilum, Methylobacterium extorquens, Aeromonas caviae, Azotobacter vinelandii, Alcaligenes latus, Pseudomonas oleovorans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas aeruginosa, Pseudomonas acidophila, Pseudomonas resinovorans, Escherichia coli und Klebsiella besteht.
  8. Bakterienstamm nach Anspruch 1, wobei das Nuclease-Gen von einem homologen Gen codiert wird, das zur Erhöhung der Nuclease-Aktivität modifiziert wurde.
  9. Bakterienstamm nach Anspruch 8, wobei der Bakterienstamm mutiert ist und die mutierten Bakterien einem Screening auf eine erhöhte Nuclease-Aktivität unterzogen werden.
  10. Bakterienstamm nach Anspruch 1, wobei der Stamm das Produkt des Nuclease-Gens von einem heterologen Nuclease-Gen exprimiert.
  11. Bakterienstamm nach Anspruch 1, wobei der Stamm das Produkt des Nuclease-Gens von einem homologen Nuclease-Gen exprimiert.
  12. Bakterienstamm nach Anspruch 1, 10 oder 11, wobei das Fermentationsprodukt ein Polyhydroxyalkanoat ist.
  13. Bakterienstamm nach Anspruch 1, 10 oder 11, wobei das Fermentationsprodukt ein Protein ist.
  14. Bakterienstamm nach Anspruch 13, wobei das Fermentationsprodukt ein intrazelluläres Protein ist.
  15. Bakterienstamm nach Anspruch 14, wobei das intrazelluläre Protein ein Enzym ist.
  16. Bakterienstamm nach Anspruch 1, wobei der Bakterienstamm gentechnisch so manipuliert ist, dass er das Fermentationsprodukt erzeugt oder dessen Erzeugung verstärkt.
  17. Bakterienstamm nach Anspruch 16, wobei das Fermentationsprodukt ein Polysaccharid ist.
  18. Bakterienstamm nach Anspruch 16, wobei das Fermentationsprodukt ein Polyhydroxyalkanoat ist.
  19. Fermentationsprozess, der das Zufügen des Bakterienstammes nach einem beliebigen der Ansprüche 1 bis 18 zu einem Wachstumsmedium umfasst.
  20. Verfahren nach Anspruch 19, wobei man den Bakterienstamm bis zu Zelldichten von wenigstens 50 g/l wachsen lässt.
  21. Verfahren nach Anspruch 19, das ferner das Lysieren der Zellen umfasst.
  22. Verfahren nach Anspruch 19, wobei der Bakterienstamm von Anspruch 3 in dem Verfahren eingesetzt wird, das ferner den Einsatz eines wässrigen Prozesses zur Herstellung einer Suspension von Poly(3-hydroxyalkanoat)-Körnchen, die im Wesentlichen frei von Nucleinsäuren ist, umfasst.
  23. Verfahren nach Anspruch 19 eingesetzt in einem Prozess zur Herstellung von Polysacchariden, die aus der Gruppe ausgewählt sind, die aus Xanthangummi, Alginaten, Gellangummi, Zooglan, Hyaluronsäure und mikrobieller Cellulose besteht
  24. Verfahren nach Anspruch 19 eingesetzt in einem Prozess zur Herstellung intrazellulärer Proteine, die aus der Gruppe ausgewählt sind, die aus Enzymen, Wachstumsfaktoren und Cytokinen besteht.
  25. Verfahren nach Anspruch 19, wobei das Bakterium eine homologe Nuclease exprimiert, das ferner das gentechnische Manipulieren der Nuclease zur Erhöhung der Nuclease-Aktivität umfasst.
EP99914271A 1998-03-30 1999-03-30 Mikrobielle stämme und verfahren für die herstellung von biomaterialien Expired - Lifetime EP1068294B1 (de)

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AU3215799A (en) 1999-10-18
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ATE386102T1 (de) 2008-03-15
CA2325350C (en) 2012-05-08
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